Bp 50-specific antibodies and fragments thereof

ABSTRACT

A new B-cell receptor, Bp50, a 50 kilodalton polypeptide, that functions in B-cell proliferation is described. Ligands such as lymphokines, antibody molecules or the Fv fragments of antibody molecules that bind to Bp50 and augment the proliferation of activated B-cells can be used to regulate B-cell proliferation or differentiation.

This is a division of U.S. Ser. No. 07/896,076, filed Jun. 2, 1992, nowU.S. Pat. No. 5,247,069; which is a division of U.S. Ser. No.07/708,075, filed May 24, 1991, now U.S. Pat. No. 5,182,368; which is acontinuation of U.S. Ser. No. 06/873,884, filed Jun. 13, 1986, nowabandoned.

1. INTRODUCTION

The present invention is directed to ligands, such as antibody moleculesor fragments of antibody molecules or other ligands such as lymphokineswhich bind to a 50 kDa B-cell surface marker, herein referred to asBp50, that functions in B-cell proliferation but not in early B-cellactivation. The present invention is also directed to the Bp50 B-cellantigen itself. In a particular embodiment of the present invention, amonoclonal antibody, G28-5, is described that defines Bp50 and appearsto play a role in the proliferation of activated B-cells but has nodetectable effect on the proliferation of resting B-cells.

The ligands, such as antibodies, lymphokines and fragments thereof ofthe present invention can be used to direct and regulate human B-cellproliferation and/or differentiation. In addition, the ligands of thepresent invention may be modified by the attachment of other compoundswhich can be used in the treatment and/or detection of malignant cellsthat express the Bp50 antigen.

2. BACKGROUND OF THE INVENTION

The activation of resting B-cells from G₀ to G₁ phase of the cell cycleand the subsequent induction of activated B-cells to proliferate aredistinct steps requiring distinct regulatory mechanisms. Some agents,including murine B-cell stimulating factor-p1 (BSF-p1) (Rabin, et al.,1985, Proc. Nat. Acad. Sci. USA 82, 2935-2939) or low doses ofanti-immunoglobulin (anti-Ig) (DeFranco, et al., 1985, J. Immunol. 135:87-94; Wetzel, et al., 1984, J. Immunol. 133: 2327-2332; DeFranco, etal., 1982, J. Exp. Med. 155: 1523-1536; Muraguchi, et al., 1984, J.Immunol. 132: 176-180), are "activation" or "competence" factors. Thatis, they induce B-cells to enlarge, synthesize more RNA, and enter G₁,but alone they do not induce DNA synthesis in B-cells. Other "growth"factors, such as human B-cell growth factor (BCGF) and interleukin-2(IL-2) cause activated B-cells to traverse the cell cycle and enter Sphase but do not trigger resting B-cells (Kehrl, et al., 1984, Immunol.Rev. 18: 75-96; Muraguchi, et al., 1984, J. Immunol. 132: 176-180;Zubler, et al. 1984, J. Exp. Med. 160: 1170-1183; Jung, et al., 1984, J.Exp. Med. 160: 1597-1604).

A number of factors that promote the growth of B-cells have now beendescribed by investigators of both murine and human systems. Theseinclude B-cell growth factors (BCGF) derived from several differentsources including T-cell lines or hybridomas, B-cell lines, or dendriticcells. Although both interleukin-1 (IL-1) and interleukin-2 (IL-2) havebeen shown to augment B-cell growth, they apparently are distinct fromcertain BCGFs. For instance, monoclonal antibodies (mAb) to a murineBCGF (O'Hara, et al., 1985, Nature (Lond.) 315: 333) or human BCGF(Ambrus, et al., 1985, J. Exp. Med. 162: 1319) block BCGF activity butnot IL-1 or IL-2 activity. Although distinct from IL-1 or IL-2, theBCGFs themselves appear to be heterogeneous based on biochemical dataand differential activity on different B-cell subsets or costimulationassays. For instance, 60-kilodalton (kDa) high-molecular-weight humanBCGF, BCGF (high), has been identified that is distinct from a 12-kDalow-molecular-weight form, BCGF (low) (Ambrus, et al., 1985, J. Clin.Invest. 75: 732). The cDNA encoding a 20-kDa murine BCGF, tentativelydesignated B-cell stimulating factor p1 (BSF-p1), has recently beencloned and sequenced (Noma et al., 1986, Nature 319: 640). Therecombinant lymphokine not only has BCGF activity but can also activateresting B-cells and induce the differentiation of IgG₁ producing cells;thus it differs from human BCGF (high) and BCGF (low) both in itsmolecular weight and in its range of activity.

These activation and growth signals presumably regulate cells byinteracting with specific B-cell surface structures. In addition to theantigen-specific signal through surface Ig, several other candidateB-cell surface polypeptides have been identified that may in some wayfunction in the activation or growth of B-cells. For instance, the cellsurface receptors for IL-1 (Dower, et al., 1985, J. Exp. Med. 160: 501)and IL-2 (Robb et al., 1984, J. Exp. Med. 160: 1126) have beencharacterized, and recently functional IL-2 receptors have beenidentified on B-cells (Zubler, et al., 1984, J. Exp. Med. 160: 1170;Jung, et al., 1984, J. Exp. Med. 160: 1597; Muraguchi, et al., 1985, J.Exp. Med. 161: 181). However, receptors for B-cell growth and activationfactors have yet to be fully characterized. Several candidate B-cellsurface polypeptides have been identified that may in some way functionin the activation or growth of B-cells. For example, Subbarao and Mosier(Subbarao, et al., 1983, Immunol. Rev. 69: 81-97) found that monoclonalantibodies (mAb) to the murine B-cell antigen Lyb2 activate B-cells, andrecently evidence has been presented suggesting Lyb2 may be the receptorfor BSF-p1 (Yakura, 1985, Fed. Proc. 44: 1532). Similarly, we have foundthat appropriate mAb (1F5) to a 35 kDa polypeptide, Bp35, activateshuman B-cells from G₀ into G₁ (Clark, et al., 1985, Proc. Nat. Acad.Sci. USA 82: 1766-1770; Gollay, et al., 1985, J. Immunol. 135:3795-3801). Aggregated C3d or antibodies to the 140 kDa C3d receptor,Bp140, cause proliferation of B-cells that are T-cell dependent(Melchers, et al., 1985, Nature 317: 264-267; Nemerow, et al., 1985, J.Immunol. 135: 3068-3073; Frade et al., 1985, Eur. J. Immunol. 15:73-76). Although BCGFs have been identified in both mouse and man, thereceptors for these factors have not yet been isolated. Wang andcoworkers (Wang, et al., 1979, J. Exp. Med. 149: 1424-1433) made apolyclonal antisera that identified a 54-kDa polypeptide (gp54) on humanB-cells and showed that the rabbit antisera to gp54 induced tonsillarB-cells to divide. Recently, Jung and Fu (Jung, et al., 1984, J. Exp.Med. 160: 1919-1924) isolated a mAb (AB-1) to a 55-kDa antigenrestricted to activated B-cells that blocks BCGF-dependentproliferation. However, whether or not either anti-gp54 or AB-1recognize a BCGF receptor is not yet known.

3. SUMMARY OF THE INVENTION

The present invention is directed to ligands which (a) bind to Bp50, a50 kDa B-cell specific surface polypeptide described herein, and (b)augment the proliferation of activated B-cells. The invention is alsodirected to the Bp50 antigen itself, which is defined by monoclonalantibody G28-5 and functions in proliferation of activated B-cells. Inaddition the invention is directed to ligands which bind to Bp50, but donot demonstrate a biological effect or function such as augmentation ofthe proliferation of activated B-cells.

The ligands of the present invention include antibody molecules,monoclonal antibody molecules and fragments of these antibody moleculeswhich contain the antigen combining site or chemically modifiedantibodies and fragments; such fragments include but are not limited tothe Fv, Fab, F(ab')₂, Fab' and the like. In addition, the ligands of thepresent invention comprise lymphokines, which can include but are notlimited to human B-cell growth factors as well as chemically modifiedlymphokines. The ligands of the present invention can be chemicallymodified, for example by linking or coupling a compound to the ligand.Such compounds include but are not limited to cytotoxic agents,therapeutic agents, chemotherapeutic agents, labels such as radiolabels,dyes, enzymes, radioopaque compounds, and the like. The ligands of thepresent invention can in their modified or unmodified form, be used todirect, regulate and modify human B-cell proliferation and/ordifferentiation.

The present invention is based upon the discovery that two human B-celldifferentiation antigens, Bp35 and the B-cell antigen described herein,Bp50, apparently play distinctive roles as signal receptors in B-cellactivation. Monoclonal antibodies (mAb) to Bp35 and Bp50 both deliverpositive signals to B-cells that stimulate their transition through thecell cycle. MAb to Bp35, like anti-Ig antibodies, functions principallyto activate resting B-cells to become competent to enter the G₁ phase ofthe cell cycle. In contrast, a monoclonal antibody described herein orits F(ab')₂ fragment to Bp50, a 50-kDa polypeptide expressed on allB-cells, functions to stimulate activated B-cells to traverse the cellcycle and augments the proliferation of activated B-cells. Monoclonalantibodies to Bp35, like anti-Ig antibodies, activate tonsillar B-cellsand induce low levels of B-cell proliferation. In contrast, anti-Bp50monoclonal antibody alone neither activates B-cells nor induces B-cellsto proliferate, but together with anti-Bp35 or anti-Ig antibodies,augments B-cell proliferation. In this respect the action of anti-Bp50antibody resembles the activity of B-cell growth factors (BCGF). Aslittle as 0.05 ug/ml of anti-Bp50 is needed to augment proliferationand, like BCGF, anti-Bp50 is effective even when added 12 to 24 hoursafter B-cells are activated with anti-Ig or anti-Bp35. Withoutadditional exogenous signals, anti-Bp35 and anti-Bp50 antibodiestogether induce strong proliferation of purified resting B-cells. Theseresults suggest that the Bp35 and Bp50 surface molecules function in theregulatory control of B-cell activation and progression through the cellcycle. Because of the significance anti-Bp35 and like molecules have onthe effect and action of the ligands of the present invention, Clark etal., 1985, Proc. Natl. Acad. Sci. (USA) 82: 1766-1770 is incorporated byreference herein.

Although the activity of anti-Bp50 resembles that of BCGF (low) sinceboth anti-Bp50 and BCGF (low) are costimulatory with the same agents butnot with each other and both anti-Bp50 and BCGF (low) affect onlyactivated B-cells and work in a soluble form, the activity of anti-Bp50can be distinguished from the activity of BCGF (low), since theproliferation of B-cells stimulated with optimal amounts of anti-Bp50and anti-Bp35 (or anti-Ig) can be augmented further with BCGF (low) andboth blood B-cells and certain B-cell lymphomas respond differently toanti-Bp50 versus BCGF. For optimal activity, anti-Bp50 should be addedwithin 12 hours of B-cell activation, whereas BCGF (low) retains optimalactivity even when added 24 hours after activation. In addition, Bp50 isexpressed on all B-cells while receptors for BCGF (low) are restrictedto activated B-cells. Thus anti-Bp50 and BCGF (low) may coordinatelyregulate B-cell growth, but apparently do so through distinct signals.

In one embodiment of the present invention, the ligands which bind toBp50 and augment the profliferation of activated B-cells can be used toincrease an immune response. For example, these ligands which bind Bp50can be used as an "adjuvant" to increase an immune response to avaccine. Alternatively, these ligands can be used to increase the immuneresponse of an immunosuppressed individual.

In another embodiment, the ligands of the invention can be chemicallymodified so that the cells to which the ligands bind are killed. Sinceall B-cells express the Bp50 antigen, this approach would result insuppression of the immune response. For example, a cytotoxic drug linkedto a ligand of the present invention can be used in vivo to causeimmunosuppresion in order to cross histocompatibility barriers intransplant patients; alternatively, these modified ligands may be usedto control autoimmune diseases.

In another embodiment of the present invention, malignancies such astumor cells that express Bp50 can be treated using a ligand of theinvention linked to a chemotherapeutic agent useful in treating suchneoplastic disease. These modified ligands can be used in vivo to directthe chemotherapeutic agent to any type of malignant cell which expressesthe Bp50 antigen including cells which are not B-cells but which doexpress Bp50. When using the ligands of the invention which augmentB-cell proliferation, a particular advantage should be realized whentreating B-cell malignancies where the chemotherapeutic agent linked tothe ligand comprises one that is more effective in killing proliferatingcells; in this instance a potentiation of the drug action should beobtained.

Alternatively, the ligands of the invention can be used in vitro toidentify or separate cells which express the Bp50 antigen and/or toassay body fluids for the presence of the Bp50 antigen which may or maynot be shed. In addition, the ligands of the invention can be used invivo in order to image cells or tumors which express the Bp50 antigen.

The purified Bp50 antigen of the present invention can be used to makeantibodies and to make or design other ligands of the invention. Inaddition the Bp50 antigen could be used in assays such as diagnosticimmunoassays. Moreover, Bp50 itself may be used as a mediator of cellimmunity in vivo or in vitro.

3.1. DEFINITIONS

As used herein, the following abbreviations will have the meaningsindicated:

AO=acridine orange

BCGF=B-cell growth factor

BCGF (high)=a 60 kDa human BCGF

BCGF (low)=a 12 kDa human BCGF

Bp35=a 35 kDa B-cell specific surface polypeptide (CD20) defined by mAb1F5

Bp50=a 50 kDa B-cell specific surface polypeptide defined by mAb G28-5

Fv=the variable region or antigen-combining site of an antibodymolecule. This may be any fragment which contains the idiotype of themolecule including but not limited to the Fab, F(ab')₂, Fab', and thelike.

IF=immunofluorescence

Ig=immunoglobulin

IL-1=interleukin 1

IL-2=interleukin 2

kDa=kilodalton

mAb=monoclonal antibody

SDS-PAGE=sodium dodecyl sulphate-polyacrylamide gel electrophoresis

TPA=12-0-tetradecanoylphorbol-13 acetate

4. DESCRIPTION OF THE FIGURES

FIG. 1. Expression of Bp50 is restricted to Bp35± B-cells. Two-colorflow cytometric analysis of 50,000 cells was performed as described(Clark, et al., 1985, Proc. Nat. Acad. Sci. USA 82: 1766-1770). The dataare plotted as cell number versus log of green fluorescence and log ofred fluorescence where 4-5 dots represent approximately a doubling offluorescence. The data are presented to show autofluorescent negativecells. PE (red) -anti-Bp35 (1F5) versus FITC (green) -anti-Bp50 (G28-5)staining shows that all Bp50+ cells are also Bp35+.

FIG. 2. Biochemical comparison of Bp50 polypeptide with other B-cellsurface antigens. Immunoprecipitation of Bp50 from surface ¹²⁵ I-labeledtonsillar cells was performed as described. Isolated antigens wereelectrophoresed on 10% SDS polyacrylamide slab gels without reduction.Gels were visualized with autoradiography and intensifying screens.Panel A: lane 1, anti-Bp50 (G28-5); lane 2, anti-Bp95 (G28-8); lane 3,sepharose-goat anti-mouse Ig only. Exposure time: 4 days. Panel B: lane1, anti-Bp50 (G28-5); lane 2, anti-Bp45 (BLAST-2); lane 3, anti-Bp39(G28-1); lane 4, anti-Bp39 (41-H16); lane 5, sepharose-goat anti-mouseIg only. An exposure time of 2 days was selected so that the bands inlanes 2 to 4 were not overexposed and could be clearly distinguishedrelative to Bp50. One of three experiments.

FIG. 3. Two-color immunofluorescence analysis of Bp50 expression.Peripheral blood or tonsillar mononuclear cells were isolated bycentrifugation on Ficoll and stained with PE (red)-conjugated G28-5(anti-Bp50) in combination with fluorescein (green)-conjugated referenceantibodies, including 2C3 (anti-IgM); 1F5 (anti-Bp35); HB10a (anti-DR);and 9.6 (anti-CD2, E receptor). Cells were analyzed with a FACS IVfitted with four decade log amplifiers in both red and green dimensions.Forward and right angle light scatter was used to gate out monocytes.Unstained cells are positioned at the back of the grid; red fluorescenceis to the right and green fluorescence is to the left.

FIG. 4. Dose response curves for augmentation of proliferation of densetonsillar Er- B-cells by anti-Bp50 antibodies as indicated: Media only;anti-Bp50 only anti-Bp35 (5 ug/ml) only; BCGF only; anti-Bp35 plus BCGF;anti-Bp35 plus graded doses of anti-Bp50. Mean proliferation±standarderror of quadruplicate samples was measured on day 3.

FIG. 5. Anti-Bp50 mAb are most effective at augmenting proliferation ifadded after a B-cell activation signal. Dense tonsillar Er- B-cells wereincubated for 4 days with media only, anti-Bp50 (0.5 ug/ml) added atdifferent times after incubation, anti-Bp35 (5 ug/ml) added at differenttimes after incubation; anti-Bp50 kept constant to which anti-Bp35 wasadded later at different times; anti-Bp35 kept constant to whichanti-Bp50 was added to cultures at different times. During the last 10hr ³ H-thymidine was added and its incorporation was measured.

FIG. 6. Comparison of the ability of anti-Bp35 and anti-Bp50 to induceresting tonsillar B-cells to leave the G₀ stage of the cell cycle. Day 3post treatment media only (₋₋₋₋₋₋₋₋₋₋), anti-Bp35 only (-- -- -- -- --);and Ig only (. . . . .), A, no additional additives; B, anti-Bp50 (0.5ug/ml) added to each group; C, 5% BCGF added to each group. Data isplotted as relative cell number versus log of AO red fluorescence (RNA).

FIG. 7. Kinetics of B-cell proliferation after stimulation withanti-Bp50 versus BCGF. Dense tonsillar E- B-cells were stimulated withmedia alone; 10% BCGF only; anti-Bp35 only; anti-Bp50 only;anti-Bp35+10% BCGF; anti-Bp35+anti-Bp50; and anti-Bp35+anti-Bp50+10%BCGF. Proliferation was measured on the days indicated by an 18-hourpulse of ³ H thymidine. Proliferation was measured in quadruplicate andstandard errors are shown. One of three experiments.

FIG. 8. Times after anti-Bp35 stimulation when anti-Bp50 (A) or BCGF (B)optimally augment proliferation. Dense tonsillar E- B-cells werestimulated as shown and proliferation was measured by an 18 hour pulseof ³ H thymidine on day 3. Media; anti-Bp35 only added at timesindicated; anti-Bp50 or BCGF only; anti-Bp35 added at start of culturefollowed by addition of anti-Bp50 or BCGF at times indicated; anti-Bp50or BCGF added at start of culture followed by anti-Bp35. One of twoexperiments. Proliferation was measured in quadruplicate and standarderrors are shown. Doses used: anti-Bp35, 5 ug/ml; anti-Bp50, 0.2 ug/nl;BCGF (low) 5%. Concentrations used were as follows: anti-Bp35, 5 ug/ml;anti-Bp50, 0.2 ug/ml; BCGF, 5%.

FIG. 9. Anti-Bp50 and BCGF have additive effects on B-cellproliferation. Dense tonsillar E- B-cells were stimulated with gradeddoses of BCGF (low) together with anti-Bp50 only; anti-Bp35 only;anti-Ig-beads only ; anti-Bp35+anti-Bp50; or anti-Bp50+anti-Ig.Proliferation was measured on day 3 after stimulation with an 18-hourpulse of ³ H thymidine. Proliferation was measured in quadruplicate andstandard errors are shown. One of four experiments. Doses used 10⁶cells: anti-Bp35, 5 ug/ml; anti-Bp50, 0.2 ug/ml; anti-Ig-beads, 50ug/ml.

FIG. 10. Comparative effects of anti-Bp50 and BCGF on normal andmalignant B-cells. Peripheral blood E- B-cells (A) or dense tonsillar E-B-cells (C) were stimulated with or without TPA (75 ng/ml) in thepresence of 10% BCGF or 1 ug/ml anti-Bp50. Two separate B-cell lymphomas(panels B and D) were stimulated in the same way. Proliferation wasmeasured on day 3 by incorporation of ³ H thymidine during a 12-hourpulse. Proliferation was measured in quadruplicate and standard errorsare shown.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to ligands which (a) bind to Bp50, a50 kDa B-cell specific surface polypeptide, and (b) augment theproliferation of activated B-cells. The invention is also directed tothe Bp50 antigen itself, which is defined by mAb G28-5 and functions inB-cell proliferation. In addition, the invention is directed to ligandswhich bind to Bp50 but do not demonstrate a biological effect orfunction such as augmentation of proliferation of activated B-cells.

The ligands of the present invention include antibody molecules,monoclonal antibody molecules and fragments of these antibody moleculeswhich contain the antigen combining site that binds to the Bp50 receptorincluding chemically modified antibodies and fragments; such fragmentsinclude but are not limited to the Fv, Fab, F(ab')₂, Fab' and the like.In addition, the ligands of the present invention comprise lymphokines,which bind to the Bp50 receptor. These may include but are not limitedto BCGFs as well as chemically modified lymphokines and the like. Theligands of the invention can be used in their modified or unmodifiedforms to modulate and regulate immune responses and in the therapy ofmalignancies which express the Bp50 antigen. These uses are discussed inmore detail in Section 5.4 below.

Where the ligand is a monoclonal antibody, or a fragment thereof, themonoclonal antibody can be prepared against Bp50 using any techniquewhich provides for the production of antibody molecules by continuouscell lines in culture. For example, the hybridoma technique originallydeveloped by Kohler and Milstein (1975, Nature 256: 495-497) as well asother techniques which have more recently become available, such as thehuman B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today4: 72) and the EBV-hybridoma technique to produce human monoclonalantibodies (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy,Alan R. Liss, Inc., pp. 77-96) and the like are within the scope of thepresent invention.

Antibody fragments which contain the idiotype of the molecule could begenerated by known techniques. For example, such fragments include butare not limited to: the F(ab')₂ fragment which can be generated bytreating the antibody molecule with pepsin; the Fab' fragments which canbe generated by reducing the disulfide bridges of the F(ab')₂ fragment;the F(ab')₂ fragment which can be generated by treating the antibodymolecule with papain; and the 2Fab or Fab fragments which can begenerated by treating the antibody molecule with papain and a reducingagent to reduce the disulfide bridges.

Where the ligand that binds Bp50 is a lymphokine, the lymphokine may beobtained from natural sources or if its amino acid sequence is known ordeduced the lymphokine can be synthesized via chemical syntheticmethods. Alternatively, if the gene sequence of the lymphokine is known,recombinant DNA techniques may be utilized to clone the gene in anexpression vector which provides for transcription and translation ofthe gene sequence in an appropriate host cell.

Depending upon its intended use, the ligand or appropriate fragments ofthe ligand may be chemically modified by the attachment of any of avariety of compounds to the ligand using coupling techniques known inthe art. Such techniques may include but are not limited to the use ofcarbodiimide, cyanogen bromide, bifunctional reagents such asglutaraldehyde, N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP),Schiff base reactions, attachment to sulfhydryl moieties, the use ofsodium isothiocyanate, or enzymatic linkage, to name but a few. Where aradioisotope is to be attached to the ligand this may also beaccomplished via enzymatic means, oxidative substitution, chelation etc.For a review of the chemical reagents which can be used for proteinmodification see, Lundblad and Noyes, Chemical Reagents for ProteinModification, Volume II, CRC Press, Inc., Boca Raton, Fla., Ch. 5,pp.123-139, 1984.

The chemical linkage or coupling of a compound to the ligand could bedirected to a site on the ligand that does not participate in binding toBp50. This could be accomplished by protecting the binding site of theligand prior to performing the coupling reaction. For example, theligand can first be bound to Bp50 in order to protect the Bp50 bindingsite, then the coupling reaction can be accomplished to link the desiredcompound to available reactive sites on the ligand-Bp50 complex. Oncethe coupling raction is complete, the complex can be disrupted therebygenerating a modified ligand to which the desired compound is attachedso that the Bp50 binding site of the molecule is minimally affected.Where the ligand comprises a monoclonal antibody such as G28-5, in whichthe Fc domain of the molecule is not required for the ligand to exertits effect (see Section 5.3.3. infra) it may be advantageous to directthe coupling of desired compounds to the Fc domain of the molecule.

The subsections below describe the new, 50-kDa B-cell surface marker,Bp50, which apparently functions in B-cell proliferation as well asligands which bind to the new 50 kDa receptor, and their uses. As anexample of the ligands of the present invention a monoclonal antibodywhich defines Bp50 and its F(ab')₂ fragments are also described which,like BCGF, augments B-cell proliferation. Unlike anti-Bp35 mAb, whichcan induce resting B-cells in G₀ to enter G₁, anti-Bp50 mAb does notactivate resting B-cells. Anti-Bp35 and anti-Bp50 mAb together, withoutany additional exogenous signals, induce strong activation andproliferation of purified B-cells.

The experiments described below also demonstrate that anti-Bp50 activityresembles BCGF activity but that anti-Bp50 is distinct from one BCGFsince anti-Bp50 and low molecular weight BCGF are clearly additive andact differently on various B-cell subsets or malignancies. Bp50 may be areceptor for a distinct BCGF or for a transmembrane signal thatmodulates BCGF production or BCGF receptor expression.

5.1. METHODS USED TO CHARACTERIZE THE Bp50 RECEPTOR Cell Preparations

Mononuclear cells were isolated from normal or leukemic heparanizedperipheral blood by Ficoll-Hypaque gradients (Pharmacia, Piscataway,N.J.). Mononuclear cells were obtained from tonsillar tissues asdescribed (Clark, et al., 1985, Proc. Nat. Acad. Sci. USA 82:1766-1770). T cells were depleted with AET-treated sheep erythrocyteresetting and Ficoll-Hypaque gradient separation. In some experimentsblood B-cells were enriched by isolating nylon wool adherent cells.Monocytes were removed by incubation on plastic petri dishes one or twotimes at 37° C. for 45 minutes unless otherwise stated. Buoyant or densetonsillar B-cell fractions were isolated by Percoll step gradients asdescribed (Clark, et al., 1985, Proc. Nat. Acad. Sci. USA 82:1766-1770). Dense tonsillar B-cell preparations consistently had greaterthan 95% sIg+ Bp35+ cells. Blood B-cell-enriched preparations had 60-85%sIg+ cells. B-cell lymphoma cells were isolated by gently teasinglymphoma cells into medium followed by Ficoll-Hypaque gradientcentrifugation.

Monoclonal Antibodies

The G28-5 antibody to Bp50 was generated by immunizing BALB/c mice withhuman E- tonsillar lymphocytes and fusing immune spleen cells with theNS-1 myeloma (Kohler, et al., 1975, Nature 256: 495-497; Ledbetter, etal., 1979, Immunol. Rev. 47: 63-82). Hybrid cell cultures secretingantibody reactive with tonsillar B-cells and not with T cells wereidentified by the use of indirect immunofluorescence (IF) and analysiswith a FACS IV cell sorter; cultures with antibody giving histogrampatterns similar to known mAb to pan B-cell markers (e.g., Bp35) werecloned and selected for further study. The G28-5 clone produced an IgG₁mAb that reacted only with normal or malignant B-cells or B-cell lines.Other mAb used in this study have been described in detail (Clark, etal., 1985, Proc. Nat. Acad. Sci. USA 82: 1766-1770; Clark et al., 1986,Human Immunol. 16: 100; Ledbetter, et al., 1986, Human Immunol. 15:30-44; Ledbetter, et al., 1985, in Perspectives in Immunogenetics andHistocompatibility, ASHI, New York, 6, pp. 325-340). These include 1F5(IgG_(2a)) anti-Bp35, HB10a (IgG_(2a)), anti-HLA-DR, 2C3 (IgG₁) anti-uchain, G19-4 (IgG₁) anti-CD3, FC-2 (IgG2a) anti-Fc receptor CD16, and9.6 (IgG_(2a)) anti-CD2 (E receptor) provided by Dr. Paul Martin(Martin, et al., 1983, J. Immunol. 131: 180). The IgG₁ mAbs werepurified by precipitation using 45% or 50% saturated ammonium sulfateand DEAE Sephacryl column chromatography, and the IgG_(2a) mAbs werepurified by the use of protein A Sepharose columns. The F(ab')₂fragments of G28-5 were prepared by the method of Parham (Parham, etal., 1983, J. Immunol. 131: 2895) purified on a 2-meter long sephacrylS200 column, and assayed for purity by SDS-PAGE (Ledbetter, et al.,1985, J. Immunol. 135: 1819). The 2C3 mAb to unchains was conjugated toSepharose 4B beads (Pharmacia Fine Chemicals, Uppsala, Sweden) usingcyanogen bromide coupling.

Fluorescein and Phycoerythrin Conjugations

Purified mAb were either directly conjugated with fluorescein usingfluorescein-isothiocyanate (FITC; Molecular Probes) (green) by themethod of Goding (Goding, et al., 1976, J. Immunol. Meth. 13: 215-226),or conjugated to R-phycoerythrin (PE) (red) by using SPDP (Pharmacia)with a method we have detailed in Ledbetter, et al., 1985, inPerspectives in Immunogenetics and Histocompatibiity, ASHI, New York, 6,119-129. Lymphoid cells were incubated in round-bottom microtiter platesfor 30 minutes with an appropriate dilution of green and/or red mAb,washed twice, and then analyzed on a FACS IV cell sorter.

Two-color Immunofluorescence

Two-color studies were done with a fluorescence-activated cell sorter(FACS IV: Becton-Dickinson, Mountain View, Calif.) by using a 560-nmdichroic mirror to split the beam and a 580 long-pass filter and a 540short-pass filter (Ditric Optics, Hudson, Mass.) in front of the red andgreen photomultiplier tubes, respectively. In addition, a two-colorcompensator (T. Nozaki, Stanford University) was used to correct forminor spillover of green and red signals. For each two-color stain, datafrom 40,000 cells were collected and stored on floppy disks. Data arepresented as cell number (vertical) versus log green fluorescence versuslog red fluorescence on a 64×64 dot grid. Approximately 4.5 dotsrepresents a doubling of fluorescence. Unstained cells are positioned atthe back corner of the grid; red fluorescence is to the right and greenfluorescence is to the left. Our flow cytometry system for two-color IFwith fluorescein and phycoerythrin is described in more detail(Ledbetter, et al., 1985, in Perspectives in Immunogenetics andHistocompatibiity, ASHI, New York, 6, 119-129 and 325-340).

Cell Culture

Blood or tonsillar lymphoid cells were cultured at 5-10×10⁵ ml inquadruplicate in 96-well microtiter plates containing 200 ul RPM1-1640medium supplemented with 15% fetal bovine serum, antibiotics, glutamine,and pyruvate (R15). After 1 to 7 days, cells were pulsed with 0.5 uCi of³ H thymidine per well (New England Nuclear, 6.7 Ci/mmol; 1 Ci=37) for18 hours. Cells were then harvested onto glass-fiber filters with a cellharvester, and radioactivity was measured in a scintillation counter. Insome experiments, antibodies or factors were added at various timesafter the start of cultures; proliferation in these experiments wasmeasured on day 3.

Costimulatory Factors

Purified BCGF was purchased from Cytokine Technology (Buffalo, New York)and contained no detectable IL-1, IL-2, or interferon activity. ThisBCGF was prepared by the method of Maizel and coworkers (Maizel, et al.,1982, Proc. Nat. Acad. Sci. USA 79: 5998), who have shown that the majorBCGF activity in this material resides in a 12-kDa species, hereinafterreferred to as "BCGF (low)" (Mehta, et al., 1985, J. Immunol. 135:3298). The purification steps included preparative scale DEAE affinitychromatography followed by hydroxylapatite column chromatography. IL-1purified to homogeneity was the generous gift of Dr. Steven Dower(Dower, et al., 1985, J. Exp. Med. 162: 501). Recombinant IL-2 waskindly provided by Cetus Corporation. TPA (12-0-tetradeconoyl phorbol13-acetate) was purchased from Sigma.

Detection of Cell Activation

Changes in cell volume induced by mAb and/or factors were measured usinga cell sorter and forward angle light-scatter. Cell cycle changes incellular RNA and DNA levels were measured by staining activated cellswith acridine orange and measuring relative cellular RNA (red) and DNA(green) content with a cell sorter by the method of Darzynkiewicz et al.(Darzynkiewicz, et al., 1980, Proc. Nat. Acad. Sci. USA 77: 6697-6702).Changes in relative levels of cell surface antigens were monitored byuse of mAb directly conjugated with fluorescein and then quantitated bydirect IF fluorescence levels with an Epics V cell sorter.

Biochemical Characterization of Bp50

Immunoprecipitation of Bp50 from surface I-labeled tonsillar cells wasperformed as described (Ledbetter, et al., 1985, J. Immunol. 134:4250-4254). Isolated antigens were electrophoresed on 10% SDSpolyacrylamide slab gels without reduction. Gels were visualized usingautoradiography at -70 ° C. and Cronex lightening plus intensifyingscreens (Dupont).

5.2. CHARACTERIZATION OF THE Bp50 RECEPTOR

The subsections below describe the results of the experiments conductedusing the methods described above.

5.2.1. IDENTIFICATION OF A B-CELL SPECIFIC 50 kDA CELL SURFACE MARKER,Bp50

A mAb to Bp50 was raised by immunizing BALB/c mice with human tonsillarlymphocytes and fusing immune spleen cells with the NS-1 myeloma. Oneclone, G28-5, produced an IgG₁ mAb that did not contain the NS-1 lightchain. Upon scrutiny by IF analysis, G28-5 was found to react only withnormal or malignant B-cells or B-cell lines. A comprehensive screeningof normal tissues by established methods (Clark, et al., 1985, Proc.Nat. Acad. Sci. USA 82: 1766-1770; Ledbetter et al., 1986, HumanImmunol.15: 30-44; Ledbetter, 1985, in Perspectives in Immunogeneticsand Histocompatibility, ASHI, New York, 6, pp. 325-340) revealed thatthe G28-5 antibody reacts with E rosette negative (Er-) cells from bloodor tonsils but not with nylon wool nonadherent T cells, PHA-inducedT-cell blasts, or with blood granulocytes, monocytes, red cells, orplatelets. It reacted strongly with all seven B lymphoblastoid celllines tested and with three Burkitt's lymphoma lines (Raji, Daudi,Namalwa), but not with four T cell lines (CEM, HSB-2, JURKAT, andHPB-ALL). All chronic lymphocytic leukemias tested (3/3) and 90% (9/10)of B lymphomas tested expressed the Bp50 marker while only 28% (2/7) ofnon T, non B CALLA⁺ acute lymphocytic leukemias expressed Bp50.

The restricted distribution of Bp50 on normal tissues was furtherconfirmed by quantitative two-color immunofluorescense (two color IF)analyses. Using an R-phycoerythrin (PE)-conjugated antibody (red) to thepan B-cell antigen Bp35 (B1, CD20) and fluorescein-conjugated anti-Bp50antibody (green), we found that Bp50 was expressed only on Bp35+ B-cells(FIG. 1) in blood or tonsils. Blood B-cells consistently expressedsomewhat lower levels of Bp50 than tonsillar B-cells; this is similar toHLA-DR expression, (Ledbetter et al., 1986, Human Immunol. 15: 30-44)and to gp54 expression (Wang, et al., 1979, J. Exp. Med. 149: 1424-1433)which are also lower on blood B-cells. Bp50 was expressed at similarlevels on tonsillar B-cell subpopulations separated on Percoll gradientsinto buoyant and dense fractions. Using our PE-conjugated mAb to the Tcell marker, CD3(T3), and NK cell-associated marker, CD16(Fc receptor)(Ledbetter, et al., 1979, Immunol. Rev. 47: 63-82), we found that Bp50is not expressed on T cells or NK cells. Using two-color IF, we alsofound that CD3+ PHA blasts that expressed high levels of IL-2 receptorsdid not express Bp50.

The G28-5 antibody reacted with a single polypeptide on tonsillarlymphocytes that migrated at approximately 50 Kd under non-reducingconditions (FIG. 2A). This molecule is larger than previously reportedB-cell markers in the same molecular weight range such as Bp39 or Bp45(Zipf, et al., 1983, J. Immunol. 131: 3064-3072; Kitner, et al., 1981,Nature 294, 458-460; Clark, et al., 1986, in Leukocyte Typing II, eds.Reinherz, et al., Springer Verlag, Berlin, Chap. 12 Vol. 2, 155-167;Slovin, et al., 1982, Proc. Nat. Acad. Sci. USA 79: 2649-2653;Thorley-Lawson, et al., 1985, J. Immunol. 134: 3007-3012, and FIG. 2B).The exposure time for this gel was selected so that the molecularweights of the other B-cell markers could be readily compared with Bp50.The Bp39 marker, unlike Bp50, is expressed on granulocytes and Bp45,unlike Bp50, is restricted to B-cell blasts. Antibodies to Bp39 (41-H16)and Bp45 (MNM6, Blast-1, Blast-2) made available through aninternational workshop (Clark, et al., 1986, in Leukocyte Typing II,eds. Reinherz, et al., Springer Verlag, Berlin, Chap. 12 Vol. 2,155-167) did not block the binding of fluoresceinated anti-Bp50antibodies to B-cells. Thus, based on tissue distribution, biochemicalanalysis, and blocking studies, the G28-5 monoclonal antibody recognizesa 50-Kd structure distinct from other known B-cell antigens.

5.2.2. EXPRESSION OF Bp50 IS RESTRICTED TO B-CELLS

Both hematopoietic tissue and cell-line distribution studies anddetailed two-color flow cytometric analyses revealed that Bp50 isexpressed only on B lymphocytes. As illustrated in FIG. 3, Bp50 isexpressed on a small subset of blood lymphocytes and on a largepopulation of tonsillar lymphocytes. Virtually all Bp50+ cells in bothblood and tonsils also expressed Bp35 and HLA-DR, but did not expressthe CD2 (FIG. 1) or CD3, T-cell molecules or the IgG Fc receptors thatare found on NK cells. Furthermore, ConA-activated CD3+ T-cell blastsexpressed IL-2 receptors but did not express Bp50.

Two-color flow cytometric analyses allow the quantitative measurement ofthe density relationship between two surface antigens. We previouslyshowed that the dense, resting B-cells in the mantle zone of secondaryfollicles express IgM and low levels of Bp35, whereas the buoyant,activated B-cells in the germinal center are IgM-negative and expresselevated levels of Bp35 (Ledbetter, et al., Human Immunol. 15: 30). FIG.3 shows that both IgM-positive and IgM-negative B-cell subsets expressedBp50 in equal amounts, indicating that Bp50 is expressed on both restingB-cells and B-cells activated in vivo.

5.3. AUGMENTATION OF B-CELL PROLIFERATION WITH ANTI-Bp50 ANTIBODY

As previously explained, B-cells can be activated with low doses ofanti-u chain specific antibodies. We recently found that theB-cell-specific marker Bp35 (B1), a 35-kDa polypeptide, may alsofunction in early B-cell activation: the 1F5 mAb to Bp35, like low dosesof anti-u antibody, activates B-cells to increase in cell volume and RNAcontent and to become responsive to BCGF (Clark, et al., 1985, Proc.Nat. Acad. Sci. USA 82: 1766-1770; Gollay, et al., 1985, J. Immunol.135: 3795-3801). Therefore, it was of interest to compare the effect ofanti-Bp50 mAb in the proliferation of untreated B-cells or B-cellsactivated with either anti-Bp35 or anti-u antibodies (Table 1).Anti-Bp35 in solution or anti-u antibodies attached to Sepharose beads,under appropriate conditions alone, could stimulate some B-cellproliferation (Table 1, line 1); in contrast, anti-Bp50 antibodies alonedid not stimulate proliferation (Table 1, line 2). However, anti-Bp50mAb augmented proliferation considerably when cultured with anti-u beadsor with anti-Bp35. In this respect anti-Bp50 resembled BCGF (Table 1,line 3). Thus, it was important to determine whether anti-Bp50 and BCGFtogether could induce B-cell proliferation. As illustrated in Table 1,line 4, anti-Bp50 and BCGF together induced no proliferation, but didaugment proliferation of either anti-u or anti-Bp35 activated cellssomewhat more than either stimulant alone. BCGF over a three-log range,when used with anti-Bp50 without other signals, had no effect onproliferation of dense B-cells even when anti-Bp50 was used at dosesranging from 0.1 to 10 ug/ml.

                  TABLE 1                                                         ______________________________________                                        Augmentation of Anti-Ig or Anti-Bp35                                          Induced B Cell Proliferation With Anti-Bp50 Antibodies                        Mean Proliferation ± S.E. of B Cells Cultured With:                        Line  Co-stimulant                                                                            Media     Anti-u-beads                                                                           Anti-Bp35                                  ______________________________________                                        1     None      1,212 ± 547                                                                          10,219 ± 462                                                                         5,539 ± 308                            2     Anti-Bp50   719 ± 718                                                                          38,792 ± 1,329                                                                      25,465 ± 616                            3     BCGF        456 ± 217                                                                          14,217 ± 445                                                                         9,443 ± 343                            4     Anti-Bp50 +                                                                             1,456 ± 126                                                                          54,393 ± 2,537                                                                        46,488 ± 3,387                              BCGF                                                                    ______________________________________                                         Proliferation of dense Er tonsillar Bcells (95% surface IgM.sup.+  cells)     was measured on day 3 as described. Briefly, 2 × 10.sup.5 cells/200     ul well were cultured in quadruplicate for 48 hrs with RPMI 1640 medium       containing 15% fetal bovine serum plus additives without antibody or with     either 2C3 monoclonal antibody to u chains coupled to sepharose beads         ("antiu beads," 50 ug/ml) or free 1F5 antiBp35 antibody (5 ug/ml).            Cultures containing media, "antiu  beads," or antiBp35 were cultured alon     or with BCGF (5% final concentration, Cytokine Technology, Buffalo, New       York; has no detectable IL1 or IL2 activity), with antiBp50 (1:1000           dilution of ascites) as costimulants. After 40 hrs cells were pulsed with     .sup.3 Hthymidine, and counts incorporated were measured after 18 hrs.   

5.3.1. ANTI-Bp50 mAb AUGMENTS PROLIFERATION ONLY AFTER B-CELLS AREACTIVATED BY ANTI-Bp35 OR ANTI-u-ANTIBODIES

The results in Table 1 suggest that anti-Bp50 mAb could not induceproliferation by itself. As shown in FIG. 4, doses of anti-Bp50 rangingfrom 0.05 ug to 2.0 ug/ml had no effect on ³ H-thymidine uptake.However, in the presence of optimal levels of anti-Bp35 mAb, as littleas 0.1 to 0.5 ug/ml of anti-Bp50 antibodies augmented proliferationsubstantially. As much as 50,000 to 70,000 cpm were detectable at theoptimal time of proliferation when highly purified B-cells were culturedonly with anti-Bp35 plus anti-Bp50. A consistent observation was thathigher doses of anti-Bp50 (greater than 2-5 ug/ml) were less effectivethan doses in the 100-200 ng range.

These results suggested that anti-Bp50 may function only after B-cellsare activated by other signals. Data shown in FIG. 5 suggest that thisis indeed the case. If B-cells were first activated with anti-Bp35,anti-Bp50 could be added as late as 24-48 hours later and still augmentproliferation at day 4. In contrast, when cells were first treated withanti-Bp50, anti-Bp35 was effective only if added within a few hoursafter the start of cultures. Similar results were found when anti-urather than anti-Bp35 was used.

5.3.2. ANTI-Bp50 mAb DO NOT ACTIVATE B-CELLS OUT OF G BUT DO INDUCEACTIVATED B-CELLS TO PROGRESS THROUGH THE CELL CYCLE

Previously, we have found that anti-Bp35, like low doses of anti-uantibodies, induce resting tonsillar B-cells in G₀ to enlarge (Clark, etal., 1986, Leukocyte Typing II, eds., Reinherz, et al., Springer Verlag,Berlin, Vol. 2, 455-462) and to enter the G₁ phase of the cell cycle(Gollay, et al., 1985, J. Immunol. 135: 3795-3801). Thus, it was ofinterest to compare the ability of anti-Bp50 mAb to anti-Bp35 mAb fortheir effects on B-cell activation. As shown in FIG. 6A, unstimulateddense tonsillar B-cells even after 3 to 4 days in culture had a uniformRNA profile characteristic of cells in G₀ (Darzynkiewicz, 1980, Proc.Nat. Acad. Sci. USA 77: 6697-6702). However, about 15-30% of cellsstimulated with anti-Bp35 or anti-u had increased RNA content indicativeof entry into G₁. In contrast, neither anti-Bp50 (FIG. 6B) nor BCGF(FIG. 6C) alone induced significant numbers of B-cells to enter G₁. Forinstance, 2 days after activation, anti-Bp35 and anti-Ig mAb inducedrespectively 13.5% and 20.9% of tonsillar cells to enter G₁, whereascells treated with only anti-Bp50 (2.7%) or BCGF (3.2%) remained atmedia control levels (2.2%). However, when either anti-Bp50 or BCGF wereadded together with anti-Bp35 or anti-u antibodies, the proportion ofcells entering G₁ increased dramatically. Similarly, anti-Bp50 and BCGFalone did not induce B-cells to enter S phase (Table 2), but togetherwith either anti-Bp35 or anti-u did increase the number of S phase cellstwo- to threefold.

                  TABLE 2                                                         ______________________________________                                        Effect of Anti-Bp50 and BCGF on                                               Cell Cycle Progression in Tonsillar Lymphocytes                               Competence Progression                                                                             % Cells                                                  Signal     Signal    G.sub.0   G.sub.1                                                                            S/G.sub.2 /M                              ______________________________________                                        media      none      89.9      7.1  2.5                                       anti-Bp35  "         80.4      14.5 3.7                                       anti-Ig    "         65.6      27.6 5.7                                       media      anti-Bp50 83.6      12.0 3.3                                       anti-Bp35  "         54.1      35.5 9.7                                       anti-Ig    "         43.6      36.2 16.2                                      media      BCGF      85.4      11.7 2.2                                       anti-Bp35  "         56.6      32.6 11.6                                      anti-Ig    "         48.4      36.1 14.1                                      ______________________________________                                         Percentage of cells in G.sub.0, G.sub.1, or S and G.sub.2 determined with     the use of the acridine orangestaining procedure (Darzynkiewicz, et al.,      1980 Proc. Nat. Acad. Sci. USA 77:6697-6702); 1 × 10.sup.6 dense        tonsillar lymphocytes with antiBp35 (5 ug/ml), antiu on beads (50 ug/ml),     antiBp50 (0.4 ug/ml), BCGF (5%) or combinations as shown.                

5.3.3. OPTIMAL CONDITIONS FOR AUGMENTING B-CELL PROLIFERATION WITHANTI-Bp50 ANTIBODIES

Antibodies to Bp50 by themselves have little or no detectable effect ondense resting B-cells (Table 3). However, in the presence of agents thatcan activate B-cells, such as anti-Ig, anti-Bp35 and TPA, anti-Bp50 mAbclearly augmented proliferation. Anti-Bp50 did not costimulate withseveral interleukins, including purified IL-1, recombinant IL-2 and BCGF(low). A comparison of the effects of anti-Bp50 with those of BCGF (low)showed that the same agents that were costimulatory with anti-Bp50 werealso costimulatory with BCGF (low) (Table 3). Of particular interest wasthe finding that together BCGF and anti-Bp50 still were notcostimulatory for resting cells.

                  TABLE 3                                                         ______________________________________                                        Augmentation of B-Cell Proliferation with                                     Anti-Bp50 Antibodies or B-Cell Growth Factor                                          Mean Proliferation ± S.E. of B-Cells                                       Cultured with:                                                                             Anti-Bp50   BCGF                                         Co-stimulant                                                                            Media      (200 ng/ml) (5%)                                         ______________________________________                                        none      96 ± 1  267 ± 15 285 ± 74                                  anti-Ig   5,833 ± 391                                                                           41,634 ± 2,103                                                                         25,094 ± 61                               anti-Bp35 457 ± 45                                                                              8,143 ± 280                                                                            1,733 ± 32                                (5 ug/ml)                                                                     TPA (2 ng/ml)                                                                           7,361 ± 537                                                                           21,163 ± 871                                                                           13,064 ± 1,030                            IL-1 (10 U/ml)                                                                          264 ± 2 308 ± 23 221 ± 8                                   IL-2 (100 U/ml)                                                                         204 ± 34                                                                              350 ± 7  220 ± 11                                  BCGF (5%) 220 ± 7 851 ± 28 270 ± 18                                  ______________________________________                                         Dense Er tonsillar Bcells (greater than 95% sIgM.sup.+  cells) cultured       for 48 hr at 2 × 10.sup.5 cells/well followed by 24 hr pulse with       .sup.3 Hthymidine before counting.                                       

The kinetics of proliferation augmented by anti-Bp50 is shown in FIG. 7.The peak of proliferation occurred at day 4 and then waned whether ornot cells were activated with anti-Bp35 or other activators such asanti-Ig or TPA. The kinetics of proliferation augmented by BCGF or byanti-Bp50 were similar.

As little as 0.05 ug of anti-Bp50 antibodies augmented proliferation. Anoptimal dose of 0.3 ug/ml was used in subsequent studies. A consistentobservation was that when using whole antibody molecules, higher dosesof anti-Bp50 (greater than 2-5 ug/ml) were less effective than doses inthe 0.1-0.5 ug/ml range.

Human B-cells are exquisitely sensitive to inhibitory effects mediatedby the Fc receptors of antibodies binding to surface Ig (Parker, 1980,Immunol. Rev. 52: 115; Bijsterbosch, et al., 1985, J. Exp. Med. 162:1825). Thus, it was important to compare the efficacy of whole anti-Bp50mAb with that of anti-Bp50 F(ab')₂ fragments. Over a 100-fold dose rangeF(ab')₂ fragments were clearly as effective as, or more effective than,whole antibody at augmenting B-cell proliferation (Table 4). Thus, theFc domain of anti-Bp50 mAb is not required for anti-Bp50 to exert itseffect and, if anything, may be inhibitory. In other words, anti-Bp50,like BCGF, apparently can act as a soluble mediator without the aid ofFc receptor-mediated accessory cell function.

                  TABLE 4                                                         ______________________________________                                        The Fc Domain of Anti-Bp50 Antibodies                                         is Not Required for Augmenting B-Cell Proliferation                                            Mean Proliferation of B Cells                                       Dose      Cultured with:                                               Anti-Bp50                                                                              (ug/ml)     Media    Anti-Bp35                                       ______________________________________                                        none     --          295 ± 16                                                                             269 ± 27                                    whole Ab 0.125       278 ± 32                                                                            5,140 ± 20                                            1.25        275 ± 24                                                                            4,686 ± 342                                           12.5        163 ± 15                                                                            3,852 ± 203                                  F(ab').sub.2                                                                           0.125       594 ± 21                                                                            10,635 ± 449                                          1.25        531 ± 3                                                                             10,893 ± 575                                          12.5        279 ± 8                                                                             9,411 ± 870                                  ______________________________________                                         Cell culture conditions were as described in Table 3.                    

5.3.4 DIFFERENCES BETWEEN ANTI-Bp50 AND BCGF (LOW) ACTIVITY

Anti-Bp50 and BCGF (low) had a similar effect on B-cells and werecostimulatory with the same agents (Table 3). However, several lines ofevidence indicate that anti-Bp50 and the BCGF used in this studyapparently operate through different signals. First, Bp50 molecules,unlike BCGF (low) receptors (Bijsterbosch, et al., 1985, J. Exp. Med.162: 1825), are expressed on resting blood B-cells (FIG. 3). Second,although both anti-Bp50 and BCGF (low) function most effectively whenadded after anti-Bp35 or anti-Ig, anti-Bp50 clearly was optimallyeffective when added 12 hours after cultures began (FIG. 8A). Incontrast, BCGF (low) could be added as long as 24 hours after start ofcultures and still optimally augment proliferation (FIG. 8B). Thesekinetic experiments, which are modeled after the approach of Howard andPaul (1983, Ann. Rev. Immunol. 1: 307), suggest that a Bp50-dependentsignal may normally exert its effect before BCGF.

Both anti-Bp50 and BCGF (low) augmented proliferation of B-cellsactivated with anti-Bp35 or anti-Ig (Table 3). However, the effect ofanti-Bp50 and BCGF (low) were additive in many experiments (FIG. 7).FIG. 9 shows a titration of BCGF (low) in an experiment where anti-Bp50was used at its optimal concentration (0.2 ug/ml). BCGF (low) couldfurther augment proliferation of resting B-cells in the presence ofanti-Bp50 after activation by either anti-Ig or by anti-Bp35. Optimalconcentrations of BCGF (low) were 5-10%, while 25% was inhibitory. Thus,when anti-Bp50 and BCGF (low) were both used at their optimalconcentrations, they still showed additive effects on B-cellproliferation.

Finally, both normal and malignant B-cell subsets differed in theirresponses to anti-Bp50 and to BCGF (low). For example, some bloodB-cells responded to BCGF (low) but did not respond to anti-Bp50 (Table5). An additional activation signal such as anti-Bp35 (Table 5) or TPA(FIG. 10) was consistently necessary to allow blood B-cells to respondto anti-Bp50. While dense tonsillar B-cells generally did not respond toeither BCGF or anti-Bp50, buoyant B-cells did respond (Table 5). B-cellmalignancies also differed in their responsiveness to anti-Bp50 versusBCGF. For example, some B-cell lymphomas responded to TPA plus BCGF(low) but not to TPA plus G28-5 anti-Bp50 (FIG. 10B and D). In contrast,dense tonsillar B-cells and peripheral blood B-cells responded to TPAplus either BCGF (low) or anti-Bp50 (FIG. 10A and C).

                                      TABLE 5                                     __________________________________________________________________________    B-Cell Subsets Differ in Their Responsiveness to Anti-Bp50 or BCGF                      Mean Proliferation (±S.E.M.) of Lymphocytes From                           Blood                  Tonsils                                      Stimulation                                                                             Exp 1  Exp 2   Exp 3   Dense  Buoyant                               __________________________________________________________________________    none      527 ± 70                                                                            659 ± 133                                                                        906 ± 106                                                                          385 ± 43                                                                            387 ± 12                         BCGF (5%) 13,918 ± 1,082                                                                      37,594 ± 2,023                                                                   3,347 ± 174                                                                        332 ± 33                                                                          1,451 ± 57                         anti-Bp50 (0.5 ug/ml)                                                                   519 ± 28                                                                          1,013 ± 81                                                                         1,151 ± 28                                                                         472 ± 8                                                                           1,543 ± 20                         anti-Bp35 (5 ug/ml)                                                                     554 ± 90                                                                            645 ± 89                                                                         665 ± 115                                                                          2,415 ± 80                                                                        1,129 ± 68                         anti-Bp50 + anti-Bp35                                                                   --     12,274 ± 546                                                                       15,667 ± 333                                                                       36,589 ± 1,335                                                                     4,843 ± 136                       anti-Bp50 + BCGF                                                                        --     --      3,943 ± 115                                                                        1,342 ± 19                                                                        2,899 ± 91                         __________________________________________________________________________     Cell culture conditions as described in Table 1. Blood Nylon wood adheren     lymphocytes (B cells plus monocytes) were depleted of monocytes by            incubation on plastic dishes overnight prior to stimulation (Exp. 1 and       Exp. 2). In Exp. 3, blood Elymphocytes were depleted of monocytes by          incubation on plastic dishes for 1 hr. prior to stimulation. Tonsillar        E.sub.r.sup.-  lymphocytes were fractionated using Percoll gradients into     dense (pellet) or buoyant (fraction 1) subsets (32).                     

5.4. USES OF ANTI-Bp50 LIGANDS AND Bp50

The ligands of the present invention may be used in vivo or in vitro, intheir unmodified or modified forms to modulate immune responses. Forexample, the ligands themselves may be used as an "adjuvant" to increasean immune response to a vaccine or to increase the immune response of animmunosuppressed individual. Alternatively, if cytotoxins oranti-proliferative agents are coupled to the ligands, these modifiedligands may be used to decrease an immune response, for example, inautoimmune disease or in transplant patients to obviate graft rejection.These modified ligands could also be used to treat malignancies thatcomprise cells or tumors which express the Bp50 antigen whether or notthe malignancy is B-cell in origin.

Both the ligands of the present invention and/or Bp50 itself can be usedin vitro. Such applications include in vitro assays, such asimmunoassays for the detection of cells which express the Bp50 antigenand/or for the detection of shed Bp50 antigen, if any, in body fluids.In this instance the ligand or Bp50 could be labeled with a radiolabel,fluor, enzyme, enzyme substrate, dye, etc. In addition, the ligands maybe used to separate and/or identify cells which express the Bp50antigen, in which case the ligand may be coupled to an immobile support,or to a fluor which can be used in a FACS (fluorescence activated cellsorter).

The various applications and uses of the ligands and Bp50 of the presentinvention are discussed in more detail below.

5.4.1. Bp50 RECEPTOR AND USES OF LIGANDS SUCH AS ANTI-Bp50 TO AUGMENTB-CELL PROLIFERATION

Previous studies have suggested that the factors involved in theinduction of B-cells from G₀ into the G₁ phase of the cell cycle aredistinct from the factors or requirements for transit into the S phase.This model is based principally on studies showing that agents such aslow doses of anti-Ig B-cell activation factors , or anti-Bp35 alone havelittle or no effect on B-cell proliferation. Yet, these same agents candrive B-cells to a point in cell activation where they are susceptibleto growth factors. In contrast, growth factors such as BCGF or IL-2alone have no effect on resting B-cells but do augment growth ofactivated B-cells.

While the present invention is not to be limited to any theory orexplanation, the results presented herein provide additional support fora model of distinct regulation of B-cell activation and growth steps.Here we have shown that activation and proliferation signals in humanB-cells may be transmitted through distinct cell surface structures.Although anti-Bp35 mAb activated B-cells to enter the G₁ phase of thecell cycle, alone, it induced little or no proliferation. Anti-Bp50 mAbhad the opposite effect: it could not activate B-cells, but when addedeven as late as 12-24 hours after activation could induce B-cell growth.

The Bp50 molecule presumably could normally function as either areceptor for a ligand such as a soluble growth factor or for a signalmediated through cell--cell contact (i.e., a ligand found on the surfaceof another cell). Previous studies have identified several Tcell-derived BCGFs that, like anti-Bp50, augment B-cell proliferation.Both high and low molecular weight forms of B-cell growth factors havebeen identified and different types have been shown to have additiveeffects (Kehrl, et al., 1984, Immunol. Rev. 18: 75-96; Kishimoto, 1985,Ann. Rev. Immunol. 3: 133-157; Swain, et al. 1983, J. Exp. Med. 158:822-835; Howard et al., 1984, Immunol. Rev. 78: 185-210; Ambrus, et al.,J. Clin. Invest. 75: 732-739; Ambrus, 1985, J. Exp. Med. 162: 1319-335).Thus, Bp50 might be a receptor for one of these actors.

With the exception of IL-2 receptors and the C3d receptor, the receptorson B-cells for growth signals have not yet been identified. The mAb AB-1reacts with a B-cell marker expressed only on activated B-cells andblocks BCGF-dependent proliferation, and thus might recognize the BCGFreceptor or a related structure. Bp50 appears to be distinct from theAB-1 marker since the AB-1 mAb does not block the binding of the G28-5anti-Bp50 antibody, and unlike the G28-5 mAb, reacts only with activatedB-cells (Jung, et al., 1984, J. Exp. Med. 160: 1919-1924). Bp50 is onall B-cells, which based on absorption analysis and direct bindingassays appears not to be the case for BCGF receptors. Our current dataindicate that Bp50 and the receptor for low molecular weight BCGF aredistinct structures. Using a rabbit heteroantiserum, Wang and coworkers(Wang, 1979, J. Exp. Med. 149: 1424-1433) previously described a 54-kDaglycoprotein, gp54, that like Bp50 is expressed on all B-cells but atlower levels on blood B-cells than tonsillar B-cells. It is possible,but unlikely, that the rabbit heteroantiserum and anti-Bp50 recognizethe same or related structures: unlike anti-Bp50 mAb, the rabbitantiserum to gp54 alone was sufficient to stimulate B-cellproliferation.

Anti-Bp35 alone, unlike anti-Bp50, can activate B-cells from G₀ to G₁and thus can be referred to as an "activation" signal. Whether or notBp35 functions only in early B-cell activation is not yet clear sinceanti-Bp35 antibodies can stimulate some B-cells to divide (Clark et al.,1985, Proc. Nat. Acad. Sci. USA 82: 1766-1770). Similarly, Bp50 may notstrictly function only as a "growth" signal: anti-Bp50 antibodiestogether with activation signals (anti-Bp35 or anti-u) not only augmentproliferation but also augment the total number of B-cells entering G₁(Table 2). In other words, anti-Bp50 as costimulant acts to promote theprogression of both the activation (G₀ to G₁) and growth (G to S) phasesof the cell cycle. The BCGF used in these studies also had similaractivity (FIG. 6C). Thus, anti-Bp35 and anti-Bp50 (or BCGF) appear to bemost analogous to the "competence" and "progression" factors describedin studies of fibroblast growth regulation. How B-cells respond toanti-Bp35 or anti-Bp50 clearly may depend on their state ofdifferentiation or activation.

Here we have shown that two mAb, anti-Bp35 (a "competence" signal) andanti-Bp50 (a "progression" signal), together can induce substantialproliferation of highly purified B-cells in the absence of antigen orother known factors. The natural ligands for these structures are notyet known. However, since mAb to appropriate epitopes can mimic bothsoluble factors and signals mediated by cell--cell interactions, it maybe possible to use appropriate combinations of mAb to direct andregulate human B-cell proliferation or differentiation. This, in turn,will help in devising strategies in vivo for the control of humandiseases such as B-cell malignancies, immunodeficiencies and certainautoimmune diseases.

The new monoclonal antibody, G28-5, that reacts with a single-chainpolypeptide of approximately 50 Kd expressed on the surface of humanB-cells is but a particular embodiment of the ligands of the presentinvention which can augment the proliferation of activated B-cells.Since human B-cell proliferation can be augmented similarly byT-cell-derived BCGFs including low- and high-molecular-weight BCGF wecompared the activity of anti-Bp50 G28-5 with that of a BCGF preparationcontaining predominantly low-molecular-weight BCGF. Anti-Bp50 G28-5 andBCGF (low) were very similar in that they were costimulatory with thesame activation agents (anti-Ig, anti-Bp35 and TPA) but were notcostimulatory with each other or with IL-1 or IL-2. Furthermore, theactivity of anti-Bp50 G28-5 was not dependent on its Fc domain sinceF(ab')₂ fragments of G28-5 were functionally active. This suggests thatsoluble anti-Bp50, like soluble BCGF, does not requireFc-receptor-bearing accessory cells to exert an effect. Furthermore,both anti-Bp50 and BCGF are effective only in the presence of anactivation stimulus. In other words, anti-Bp50 and BCGF are not"competence" factors, but rather promote the "progression" of B-cellsthrough the cell cycle.

While it is possible that Bp50 may function as receptor for a ligandsuch as a B-cell growth factor, several results suggest that Bp50 is notthe receptor at least for the BCGF (low) used in this study: it isexpressed on blood B-cells while BCGF (low) receptors apparently arenot. Candidate structures for the BCGF (low) receptor, unlike Bp50, arealso expressed only on activated B-cells. Furthermore, both normal andmalignant B-cell populations differ in their responsiveness to anti-Bp50versus BCGF (low) (Table 5 and FIG. 10). For instance, some B lymphomasproliferate in response to BCGF (low), but not in response to anti-Bp50.Finally, in a number of experiments, optimal concentrations of anti-Bp50and BCGF together induced more proliferation than either one alone.Anti-Bp50 mimics the activity of other BCGF, such as BCGF (high) thatare co-stimulatory with anti-IgM (Ambrus, et al., 1985, J. Exp. Med.162: 1319; Ambrus, et al., 1985, J. Clin. Invest. 75: 732). Thissuggests that Bp50 could function as the receptor for BCGF (high).

Although Bp50 may be a receptor for a soluble ligand, alternatively,Bp50 may function as a receptor for a cell--cell mediated signal thatregulates BCGF receptor levels and/or autocrine production. Precedencefor differentiation antigens serving as amplifiers of anautocrine-receptor pathway comes from studies with T cells. MAb to theLp220 common leukocyte antigen augments proliferation by elevating IL-2receptor expression on activated T cells (Ledbetter, et al., 1985, J.Immunol. 135: 1819). An analogous mechanism may be operating withanti-Bp50 and expression of certain BCGF receptors. Bp50 and BCGF (low)apparently are under some coordinate control since, like IL-1 and IL-2receptors, BCGF augments expression of Bp50 on certain leukemic cells.The Bp50 molecule also shares similarities with the Tp44 molecule thatfunctions to influence IL-2 production. We and others have shown thatthe 9.3 anti-Tp44 antibody augments proliferation of T cells activatedby anti-CD3 or TPA (Ledbetter, et al., 1985, J. Immunol. 135: 2331;Hara, et al., 1985, J. Exp. Med. 161: 1513). Similarly, anti-Bp50augments the proliferation of B-cells activated by anti-Bp35 or TPA. TheTp44 signal functions by stimulating IL-2 production rather than bystimulating T cell growth. The Bp50 signal presumably could function inan analogous manner by stimulating B-cell autocrine production (Gordon,et al., 1984, Nature, Lond. 310: 145).

5.4.2. MODIFIED LIGANDS USED FOR IMMUNOSUPPRESSION OR TREATMENT OFMALIGNANACIES

According to this embodiment, the ligand of the present invention can bemodified by the attachment of an antiproliferative agent so that theresulting molecule can be used to kill cells which express the Bp50antigen. Such modified ligands may be used in the treatment ofautoimmune disease in order to suppress the proliferation of B-cells andthereby suppress the autoimmune response. These modified ligands canalso be used to immunosuppress a transplant patient to prevent rejectionof a graft. Accordingly, cytotoxic agents which are used for thesuppression of immune responses can be attached to the ligands of theinvention. When using ligands which augment the proliferation ofB-cells, an increased effect should result because the drug will bedirected to proliferating B-cells.

In another embodiment, the ligands of the present invention which aremodified by the attachment of an antiproliferative agent can be used totreat malignancies in which tumors or cells express the Bp50 antigen.Attachment of these chemotherapeutic agents to the ligands of theinvention should result in a greater specificity of the drug for themalignant cells. Moreover, a particular advantage should be obtainedwhen treating a B-cell malignancy with a ligand coupled to a cytotoxinwhich is more effective in killing proliferating cells thannon-proliferating cells; treatment with such a ligand should result in apotentiation of the action of the cytotoxin.

Accordingly, the chemotherapeutic agents or antiproliferative agentswhich can be coupled to the ligands of the present invention include butare not limited to the agents listed in Table 6 below which is derivedfrom Goodman and Gilman, The Pharmacological Basis of Therapeutics,Sixth Edition, MacMillan Publishing Co., Inc, New York, pp. 1249-1313,1980 which is incorporated by reference herein.

                  TABLE 6                                                         ______________________________________                                        Chemotherapeutic Agents Which Can be Coupled to Anti-Bp50 Ligands             Class       Type           Agent                                              ______________________________________                                        Alkylating Agent                                                                          Nitrogen Mustard                                                                             Mechlorethamine                                                               Cyclophosphamide                                                              Melphalan                                                                     Uracil Mustard                                                                Chlorambucil                                                   Ethylenimine Derivatives                                                                     Thiotepa                                                       Alkyl Sulfonates                                                                             Busulfan                                                       Nitrosoureas   Carmustine                                                                    Lomustine                                                                     Semustine                                                                     Streptozocin                                                   Triazenres     Dacarbazine                                        Antimetabolites                                                                           Folic Acid Analogs                                                                           Methotrexate                                                   Pyrimidine Analogs                                                                           Fluorouracil                                                                  Cytarabine                                                                    Azaribine                                                      Purine Anlogs  Mercaptopurine                                                                Thioguanine                                        Natural Products                                                                          Vinca Alkaloids                                                                              Vinblastine                                                                   Vincristine                                                    Antibiotics    Dactinomycin                                                                  Daunorubicin                                                                  Doxorubicin                                                                   Bleomycin                                                                     Mithramycin                                                                   Mitomycin                                                      Enzymes        L-Asparaginase                                     Miscellaneous Agents                                                                      Platinum Coordinated                                                                         Cisplatin                                                      Complexes                                                                     Substituted Urea                                                                             Hydroxyurea                                                    Methyl Hydrazine                                                                             Procarbazine                                                   Derivative                                                                    Adrenocortical Mitotane                                                       Suppressant                                                       Hormones and                                                                              Adrenocorticosteroids                                                                        Prednisone                                         Antagonists                                                                               Progestins     Hydroxyprogesterone                                                           caproate                                                                      Medroprogesterone                                                             acetate                                                                       Megestrol acetate                                              Estrogens      Diethylstilbestrol                                                            Ethinyl estradiol                                              Antiestrogen   Tamoxifen                                                      Androgens      Testosterone                                                                  propionate                                                                    Fluoxymesterone                                    Radioactive Isotopes                                                                      Phosphorous    Sodium phosphate .sup.32 P                                     Iodine         Sodium Iodide .sup.131 I                           ______________________________________                                    

Any method known in the art can be used to couple the ligand to thechemotherapeutic or antiproliferative agent. Examples of such methodshave been enumerated previously (see Section 5, supra).

5.4.3. OTHER USES OF LIGANDS AND Bp50

In addition to the therapeutic applications the ligands and Bp50 itselfhave other applications in both in vitro and in vitro diagnostic assays,separation schemes, etc.

The Bp50 receptor can be used to manufacture and/or design the ligandsof the invention. Bp50 can also be used with the ligands of theinvention in assays in vitro which require a standard to quantify theamount of Bp50 detected in a sample. Ultimately, Bp50, itself may beuseful as a soluble factor which mediates immunity, e.g. a lymphokine.

In addition to therapeutic treatment and diagnostic assays, the ligandsof the present invention could be used for identifying or separatingcells which express the Bp50 antigen. In addition, if an appropriateradiolabel or radio-opaque compound is linked to the ligand, the ligandcould be used for in vivo imaging of tumors which express the Bp50antigen. Other uses should become apparent to those skilled in the artfrom the foregoing description.

6. DEPOSIT OF CELL LINES

The following hybridoma has been deposited with the American TypeCulture Collection, Rockville, Md. 20852, on May 22, 1986, and has beenassigned the listed accession number:

    ______________________________________                                        Hybridoma   ATCC Accession Number                                             ______________________________________                                        G28-5       HB9110                                                            ______________________________________                                    

The present invention is not to be limited in scope by the hybridomadeposited since the deposited embodiment is intended as a singleillustration of one aspect of the invention and any cell lines which arefunctionally equivalent are within the scope of this invention. Indeedvarious modifications of the invention in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description and accompanying drawings. Such modificationsare intended to fall within the scope of the appended claims.

What is claimed is:
 1. A substantially pure antibody molecule or afragment thereof that(a) binds to Bp50, a 50 kilodalton B-cell surfaceantigen defined by monoclonal antibody G28-5, produced by a hybridomacell line deposited with the ATCC having accession number HB9110; and(b) upon binding to BP50 expressed on an activated B-cell, augmentsproliferation of the activated B-cell in a dose range of 0.1 to 0.5ug/ml, as measured by in vitro ³ H!-thymdine uptake.
 2. The antibodymolecule of claim 1 in which the fragment contains an Fv, Fab, F(ab')2or Fab' portion of the antibody.